22 Determination of Available Lysine by Various Procedures in Maillard-Type Products 1
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H. F. ERBERSDOBLER and T. R. ANDERSON Institut für Physiologie, Physiologische Chemie und Ernährungs-physiologie der Tierärztlichen Fakultät der Ludwig-Maximilians-Universität München, Veterinärstrasse 13, D 8000 München, Federal Republic of Germany Three methods of reactive-lysine determination employing the reagents Acid Orange 12, succinic anhydride, and dansyl chloride, as well as methods for total lysine and furosine using ion exchange chromatography, were compared with the method for fluorodinitrobenzene(FDNB)-reactive lysine as reference, in their a b i l i t y to estimate available lysine in heat-damaged soya protein samples. A l l methods showed at least some measure of sensitivity to increased severity of lysine damage, with samples heated alone being least damaged followed by those heated with lactose, glucose, then xylose. Under more severe conditions lactose showed reactivity comparable to that of xylose, probably owing to partial hydrolysis of the disaccharide. Total lysine overestimated FDNB lysine at a l l levels of heat damage. Acid Orange 12 and succinic anhydride values correlated well with FDNB lysine for both non-Maillard ( r = 0.93 ) and Maillard ( r = 0.90 ) material. The dansyl method gave good relative values but certain problems prevented exact quantitative calculations. The chemical methods correlated quite well with plasma lysine and lysine d i g e s t i b i l i t y methods although the absolute values varied considerably between methods.
The n u t r i t i o n a l a v a i l a b i l i t y of l y s i n e i n foods of p l a n t and animal o r i g i n may be s i g n i f i c a n t l y decreased by the ready i n v o l v e ment of the €~NH2 group of l y s i n e i n i n t r a - and intermolecular 1
Current address: Department of Human Nutrition and Food Science, University of Kiel, Dusternbrooker Weg 17-19, D 2300 Kiel, Federal Republic of Germany Current address: Biochemistry Department, University of Zululand, Private Bag X1001, Kwa-Dlangezwa 3886, South Africa 2
0097-6156/83/0215-0419$06.00/0 © 1983 American Chemical Society
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MAILLARD REACTIONS
c r o s s l i n k i n g i n p r o t e i n s as w e l l as i n r e a c t i o n s with v a r i o u s food c o n s t i t u e n t s . The most common involvement i s that together with reducing sugars i n the M a i l l a r d condensation since t h i s r e a c t i o n occurs a l s o under mild and moderate c o n d i t i o n s such as storage of the food. Some of the intermediate or end products have been repor ted to be t o x i c ( J_, 2_> 3_ ) . Although the f i n a l answer as to the n u t r i t i o n a l consequences of processing damage to l y s i n e can o b v i ously be given only by i n v i v o a n a l y s i s , r e c e n t l y emphasis has been placed on methods that not only give an acceptable estimate of a v a i l a b l e l y s i n e but are simple and r a p i d enough to be automated f o r use i n general laboratory p r a c t i c e ( e.g.4_, .5 ) . In t h i s report various chemical and i n v i v o methods are com pared i n t h e i r a b i l i t y to assess n u t r i t i o n a l damage i n v a r i o u s model soya p r o t e i n s representing mainly the M a i l l a r d - t y p e m o d i f i c a t i o n of l y s i n e . M a t e r i a l s and Methods Model p r o t e i n samples. I s o l a t e d soya p r o t e i n (Purina Brand Assay P r o t e i n RP 100 ) was employed i n these studies and found to contain on a dry matter b a s i s 96% crude p r o t e i n , 1.4% ash, 0.2% crude fiber, and 2% N-free e x t r a c t . This m a t e r i a l was used to prepare 48 samples, which were heated i n a i r t i g h t metal containers ( 250-ml ) at temperatures of 90, 110, 130° C f o r 0.5, 1, 2, and 4 h respectively. The samples were prepared by a mixture of 90 parts of soya p r o t e i n with 10 parts of tap water or of 80 parts of soya p r o t e i n with 10 parts of glucose and 10 parts of tap water or instead of glucose an equivalent amount of l a c t o s e or xylose according to t h e i r molecular mass compared on water-free basis to the glucose. In t h i s way f o r instance the l a c t o s e samples contained 71 g soya p r o t e i n , 19 g l a c t o s e and 10 g tap water. A f u r t h e r 3 samples were prepared by heating equal masses of i s o l a t e d soya p r o t e i n and tap water i n open v e s s e l s f o r 24 h at e i t h e r 95, 138, or 160° C. Crude p r o t e i n and t o t a l l y s i n e plus f u r o s i n e Total n i t r o gen was determined by macro K j e l d a h l d i g e s t i o n and p r o t e i n estima ted as Ν χ 6.25. T o t a l l y s i n e values were obtained from conventional amino a c i d analyses c a r r i e d out on 500-mg samples f o l l o w i n g d i g e s t i o n with 800 ml of 6M HC1 under r e f l u x by use o f a B i o t r o n i c LC 6000 or Kontron Liquimat I I I amino a c i d analyzer. Furosine was determined i n the same way using 300 ml 7.8 M HC1 as described i n ( 6_ ) with an amino a c i d analyzer ( 1_ ). Fluorodinitrobenzene ( FDNB ) - r e a c t i v e l y s i n e The d i r e c t FDNB method of Carpenter ( 8^ ) was used with some f u r t h e r ( 9_, _K) ) modifications.
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ERBERSDOBLER A N D ANDERSON
Determination of Lysine
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S u c c i n i c anhydride ( S A ) - r e a c t i v e l y s i n e The method of Anderson ( _Π_ ) was employed. D u p l i c a t e 12- to 15- mg p o r t i o n s of each sample ( p a r t i c l e s i z e < 8 0 / i m ) were a c c u r a t e l y weighed i n t o 20-ml glass s c i n t i l l a t i o n v i a l s , 3 ml o f 6M guanidine hydrochloride and 0.1 ml o f 5M NaOH added, and the whole was m a g n e t i c a l l y s t i r r e d at ca. 50 C f o r 15 min. A f t e r c o a l i n g to ca. 25 C, the suspension was treated with s o l i d [1,4- C] - s u c c i n i c anhydride with a s p e c i f i c a c t i v i t y of 0.026 u C i per jimol ( Radiochemical Centre, Amersham, England ) . The reagent was added i n small por t i o n s over a p e r i o d of 30 min to give an approximately 8 0 - f o l d molar excess over t o t a l l y s i n e content. Vigorous s t i r r i n g was maintained throughout the a d d i t i o n process during which a f u r t h e r two a l i q u o t s ( 0.1 and 0.05 ml ) of 5M NaOH were added a f t e r c a . 10 and 20 min r e s p e c t i v e l y . The mixture was then treated with 5 ml of a l k a l i n e h y d r o x y l amine reagent ( 20 ml hydroxylamine h y d r o c h l o r i d e t i t r a t e d to pH 13 with 3.5M NaOH ) f o r 5 min and the p r o t e i n p r e c i p i t a t e d with 5% t r i c h l o r o a c e t i c a c i d . A f t e r c e n t r i f u g i n g ( 5000 g ) f o r 5 min the supernatant was c a r e f u l l y decanted and the p r e c i p i t a t e washed twice with 10-ml a l i q u o t s of absolute ethanol. The m a t e r i a l was then d i ssolved i n 1 ml, o f 0.2M NaOH, mixed with 10 ml o f I n s t a — gel s c i n t i l l a t o r ( Packard Instrument (Pty) Ltd.) , and the r a d i o a c t i v i t y counted i n a Packard T r i Carb L i q u i d S c i n t i l l a t i o n Spectrometer. Dansyl c h l o r i d e ( DAN ) - r e a c t i v e l y s i n e The method of C h r i s t o f f e r s ( j_2 ) was employed. D u p l i c a t e 19- to 21-mg p o r t i o n s of each sample ( p a r t i c l e s i z e
